A Targetron-Recombinase System for Large-Scale Genome Engineering of Clostridia

ABSTRACT Clostridia are a group of Gram-positive anaerobic bacteria of medical and industrial importance for which limited genetic methods are available. Here, we demonstrate an approach to make large genomic deletions and insertions in the model Clostridium phytofermentans by combining designed gro...

Descripción completa

Guardado en:

Detalles Bibliográficos
Autores principales:	Tristan Cerisy, William Rostain, Audam Chhun, Magali Boutard, Marcel Salanoubat, Andrew C. Tolonen
Formato:	article
Lenguaje:	EN
Publicado:	American Society for Microbiology 2019
Materias:	clostridia engineering prophage recombinase Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/ab05c80fb10a4397b257fe22ee7054a1
Etiquetas:	Agregar Etiqueta Sin Etiquetas, Sea el primero en etiquetar este registro!

id	oai:doaj.org-article:ab05c80fb10a4397b257fe22ee7054a1
record_format	dspace
spelling	oai:doaj.org-article:ab05c80fb10a4397b257fe22ee7054a12021-11-15T15:22:24ZA Targetron-Recombinase System for Large-Scale Genome Engineering of Clostridia10.1128/mSphere.00710-192379-5042https://doaj.org/article/ab05c80fb10a4397b257fe22ee7054a12019-12-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mSphere.00710-19https://doaj.org/toc/2379-5042ABSTRACT Clostridia are a group of Gram-positive anaerobic bacteria of medical and industrial importance for which limited genetic methods are available. Here, we demonstrate an approach to make large genomic deletions and insertions in the model Clostridium phytofermentans by combining designed group II introns (targetrons) and Cre recombinase. We apply these methods to delete a 50-gene prophage island by programming targetrons to position markerless lox66 and lox71 sites, which mediate deletion of the intervening 39-kb DNA region using Cre recombinase. Gene expression and growth of the deletion strain showed that the prophage genes contribute to fitness on nonpreferred carbon sources. We also inserted an inducible fluorescent reporter gene into a neutral genomic site by recombination-mediated cassette exchange (RMCE) between genomic and plasmid-based tandem lox sites bearing heterospecific spacers to prevent intracassette recombination. These approaches generally enable facile markerless genome engineering in clostridia to study their genome structure and regulation. IMPORTANCE Clostridia are anaerobic bacteria with important roles in intestinal and soil microbiomes. The inability to experimentally modify the genomes of clostridia has limited their study and application in biotechnology. Here, we developed a targetron-recombinase system to efficiently make large targeted genomic deletions and insertions using the model Clostridium phytofermentans. We applied this approach to reveal the importance of a prophage to host fitness and introduce an inducible reporter by recombination-mediated cassette exchange.Tristan CerisyWilliam RostainAudam ChhunMagali BoutardMarcel SalanoubatAndrew C. TolonenAmerican Society for MicrobiologyarticleclostridiaengineeringprophagerecombinaseMicrobiologyQR1-502ENmSphere, Vol 4, Iss 6 (2019)
institution	DOAJ
collection	DOAJ
language	EN
topic	clostridia engineering prophage recombinase Microbiology QR1-502
spellingShingle	clostridia engineering prophage recombinase Microbiology QR1-502 Tristan Cerisy William Rostain Audam Chhun Magali Boutard Marcel Salanoubat Andrew C. Tolonen A Targetron-Recombinase System for Large-Scale Genome Engineering of Clostridia
description	ABSTRACT Clostridia are a group of Gram-positive anaerobic bacteria of medical and industrial importance for which limited genetic methods are available. Here, we demonstrate an approach to make large genomic deletions and insertions in the model Clostridium phytofermentans by combining designed group II introns (targetrons) and Cre recombinase. We apply these methods to delete a 50-gene prophage island by programming targetrons to position markerless lox66 and lox71 sites, which mediate deletion of the intervening 39-kb DNA region using Cre recombinase. Gene expression and growth of the deletion strain showed that the prophage genes contribute to fitness on nonpreferred carbon sources. We also inserted an inducible fluorescent reporter gene into a neutral genomic site by recombination-mediated cassette exchange (RMCE) between genomic and plasmid-based tandem lox sites bearing heterospecific spacers to prevent intracassette recombination. These approaches generally enable facile markerless genome engineering in clostridia to study their genome structure and regulation. IMPORTANCE Clostridia are anaerobic bacteria with important roles in intestinal and soil microbiomes. The inability to experimentally modify the genomes of clostridia has limited their study and application in biotechnology. Here, we developed a targetron-recombinase system to efficiently make large targeted genomic deletions and insertions using the model Clostridium phytofermentans. We applied this approach to reveal the importance of a prophage to host fitness and introduce an inducible reporter by recombination-mediated cassette exchange.
format	article
author	Tristan Cerisy William Rostain Audam Chhun Magali Boutard Marcel Salanoubat Andrew C. Tolonen
author_facet	Tristan Cerisy William Rostain Audam Chhun Magali Boutard Marcel Salanoubat Andrew C. Tolonen
author_sort	Tristan Cerisy
title	A Targetron-Recombinase System for Large-Scale Genome Engineering of Clostridia
title_short	A Targetron-Recombinase System for Large-Scale Genome Engineering of Clostridia
title_full	A Targetron-Recombinase System for Large-Scale Genome Engineering of Clostridia
title_fullStr	A Targetron-Recombinase System for Large-Scale Genome Engineering of Clostridia
title_full_unstemmed	A Targetron-Recombinase System for Large-Scale Genome Engineering of Clostridia
title_sort	targetron-recombinase system for large-scale genome engineering of clostridia
publisher	American Society for Microbiology
publishDate	2019
url	https://doaj.org/article/ab05c80fb10a4397b257fe22ee7054a1
work_keys_str_mv	AT tristancerisy atargetronrecombinasesystemforlargescalegenomeengineeringofclostridia AT williamrostain atargetronrecombinasesystemforlargescalegenomeengineeringofclostridia AT audamchhun atargetronrecombinasesystemforlargescalegenomeengineeringofclostridia AT magaliboutard atargetronrecombinasesystemforlargescalegenomeengineeringofclostridia AT marcelsalanoubat atargetronrecombinasesystemforlargescalegenomeengineeringofclostridia AT andrewctolonen atargetronrecombinasesystemforlargescalegenomeengineeringofclostridia AT tristancerisy targetronrecombinasesystemforlargescalegenomeengineeringofclostridia AT williamrostain targetronrecombinasesystemforlargescalegenomeengineeringofclostridia AT audamchhun targetronrecombinasesystemforlargescalegenomeengineeringofclostridia AT magaliboutard targetronrecombinasesystemforlargescalegenomeengineeringofclostridia AT marcelsalanoubat targetronrecombinasesystemforlargescalegenomeengineeringofclostridia AT andrewctolonen targetronrecombinasesystemforlargescalegenomeengineeringofclostridia
_version_	1718428024230117376

A Targetron-Recombinase System for Large-Scale Genome Engineering of Clostridia

Ejemplares similares